We acknowledge the technical assistance of the Translational Research Institute Histology core and Biological Resource Facility. This research was supported by funding from a Princess Alexandra Research Foundation award (I.V., E.D.W.), and the Medical Research Future Fund (MRFF) Rapid Applied Research Translation Program (Centre for Personalised Analysis of Cancers (CPAC; E.D.W., I.V.). The Translational Research Institute receives support from the Australian Government.

Patients have consented to this study following their admission under the Urology team at the Princess Alexandra Hospital, Brisbane, Australia. This study was performed in accordance with the principles of the Declaration of Helsinki and within ethical and institutional guidelines (ethics number HREC/05/QPAH/95, QUT 1000001165).
NOTE: As eligibility criteria, patients were aged &#8805; 18 with cancer, and able to understand and provide consent. Those who were not able to give informed consent ...

<ol>
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The authors have no conflicts of interest.

While 3D organoid protocols derived from bladder cancer tissue are still in their infancy, they are an area of active research and clinical investigation. Here, an optimized protocol to successfully establish bladder cancer PDOs that are suitable for both NMIBC and MIBC specimens is detailed. This workflow integrates parallelly into hospital-based clinical trials and considers biobank sample accrual, including histological sample processing and fresh frozen tissue banking, which is an important consideration for clinical...

Bladder cancer is the most prevalent urinary tract cancer and the tenth most common human malignancy worldwide1. It encompasses a genetically diverse and phenotypically complex spectrum of disease2. Urothelial non-muscle-invasive forms of bladder cancer (NMIBC) are the most common bladder cancer diagnoses (70%-80%), and these cancers display considerable biological heterogeneity and variable clinical outcomes2,3,4. Patients with NMIBC typically experience a high risk of disease recurrence (50-70%) and one-third of cancers will progress and develop into significantly more aggressive muscle-invasive bladder cancer (MIBC)2. Although 5-year survival rates for NMIBC are high (&#62;90%), these patients must undergo long-term clinical management5. On the other hand, locally advanced (unresectable) or metastatic MIBC is generally considered incurable6. Consequently, bladder cancer has one of the highest lifetime treatment costs within cancer care and is a significant burden for both the individual and the healthcare system3,7. The underlying genetic aberrations in advanced disease renders therapeutic management of bladder cancer a clinical challenge, and therapeutic options for invasive urothelial tumors have only recently improved since the approval of immunotherapies for both advanced and high-risk NMIBC8,9. Currently, clinical decision-making has been guided by conventional clinical and histopathological features, despite individual bladder cancer tumors showing large differences in disease aggressiveness and response to therapy10. There is an urgent need to accelerate research into clinically useful models to improve the prediction of individual patient prognosis and identification of effective treatments.
Three-dimensional (3D) organoids show great potential as tumor models due to their ability to self-organize and recapitulate the original tumor&#39;s intrinsic in vivo architecture and pharmacogenomic profile, and their capability to mirror the native cellular functionality of the original tissue from which they were derived11,12,13. Although established bladder cancer cell lines are readily available, relatively cost-effective, scalable, and simple to manipulate, the in vitro cell lines largely fail to mimic the spectrum of diverse genetic and epigenetic alterations observed in clinical bladder cancers12,14 and were all established and maintained under 2D, adherent culture conditions. Additionally, cell lines derived from primary and metastatic bladder tumors harbor significant genetic divergence from the original tumor material. 8,15.
An alternative approach is to use genetically engineered and carcinogen-induced mouse models. However, while these models recapitulate some of the natural oncogenic cascades involved in human neoplasia (reviewed in refs16,17,18), they lack tumor heterogeneity, are expensive, poorly represent invasive and metastatic bladder cancer, and are not viable for rapid term drug testing as tumors can take many months to develop14,19. Patient-derived models of cancer (including organoids, conditionally reprogrammed primary cell culture, and xenografts) provide invaluable opportunities to understand the effects of drug treatment before clinical treatment20. Despite this, few groups routinely use these patient-proximal models due to limited access to fresh primary patient tissue and the extensive optimization required to reproducibly generate patient-derived organoid (PDO) culture conditions. In an in vivo setting, oncogenic cells can interact and communicate with various compositions of the surrounding constituents, including stromal cells, tissue infiltrating immune cells, and matrix12. Similarly, for PDOs grown in a 3D format, cellular/matrix complexity can be customized to include other relevant components. PDOs can be rapidly generated and are often able to be passaged extensively or cryopreserved for later use, despite having a finite lifespan21,22,23. Pharmacodynamics (i.e., response to a drug) can be evaluated using multiple read-outs, including organoid viability and morphology, and characterization of immunohistochemistry targets or transcriptional changes.
Here, the procedures for the establishment of bladder cancer organoids from patient material collected from transurethral resection of bladder tumor (TURBT) or surgical removal of the bladder (radical cystectomy) are described. The method to generate PDOs is illustrated, using readily available wet laboratory materials and tools. Endpoints include changes in cell morphological characteristics and viability. These were measured using fluorescence microscopy, in vitro viability (metabolic and cell membrane integrity) assays, and histopathological analysis. Figure 1 shows the workflow for establishing human bladder cancer PDOs from clinical material obtained during elective surgery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.2 mL cryogenic vial</td>
			<td>Corning</td>
			<td>430487</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>Sigma-Aldrich</td>
			<td>T9661-500EA</td>
			<td>polypropylene single-use tube</td>
		</tr>
		<tr>
			<td>100 &#181;M reversible strainer</td>
			<td>STEMCELL Technologies</td>
			<td>#27270</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Collagenase/ Hyaluronidase</td>
			<td>STEMCELL Technologies</td>
			<td>#07912</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#181;M reversible strainer</td>
			<td>STEMCELL Technologies</td>
			<td>#27250</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL falcon tube</td>
			<td>Corning</td>
			<td>CLS430829-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>70% (w/w) ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-80&#176;C freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well ultra low attachment plate (Black)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3474-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>BioScientific</td>
			<td>2939</td>
			<td>Prevents the growth-inhibitory effects of TGF-&#946;</td>
		</tr>
		<tr>
			<td>ACK lysis buffer</td>
			<td>STEMCELL Technologies</td>
			<td>#07850</td>
			<td></td>
		</tr>
		<tr>
			<td>adDMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634028</td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>Animal-free recombinant EGF</td>
			<td>Sigma</td>
			<td>518179</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>Automated cell counter (TC20)</td>
			<td>Bio-rad</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 additive</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>Increases sphere-forming efficiency</td>
		</tr>
		<tr>
			<td>Cell Counting Slides</td>
			<td>Bio-rad</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>EP022628188</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>STEMCELL Technologies</td>
			<td>#07930</td>
			<td>Cell freezing solution</td>
		</tr>
		<tr>
			<td>Dispase II, powder</td>
			<td>Thermofisher</td>
			<td>17105041</td>
			<td>To enzymatically disrupt Matrigel</td>
		</tr>
		<tr>
			<td>DNAse 1</td>
			<td>STEMCELL Technologies</td>
			<td>#07900</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Thermofisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry and wet ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Esky</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Farmdyne (Iodine 16g/L)</td>
			<td>Ecolab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered, 10%</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td>Histological tissue fixative</td>
		</tr>
		<tr>
			<td>Glutamax (L-alanine-L-glutamine)</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>Source of nitrogen for the synthesis of proteins, nucleic acids</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>All-purpose buffer</td>
		</tr>
		<tr>
			<td>Histology cassette</td>
			<td>ProSciTech</td>
			<td>RCH44-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-10</td>
			<td>Peprotech</td>
			<td>100-26-25</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>Human FGF-2</td>
			<td>Peprotech</td>
			<td>100-18B-50</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (Growth Factor Reduced (GFR), phenol red-free, LDEV free)</td>
			<td>In Vitro Technologies</td>
			<td>356231</td>
			<td>Basement membrane extract (BME)</td>
		</tr>
		<tr>
			<td>Mr. Frosty freezing container</td>
			<td>ThermoFisher</td>
			<td>5100-0001</td>
			<td>cell freezing container</td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine (NAC)</td>
			<td>Sigma</td>
			<td>A7250</td>
			<td>Anti-oxidant required to protect against ROS-induced cytotoxicity</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636-100G</td>
			<td>SIRT-1 inhibitor</td>
		</tr>
		<tr>
			<td>NikonTs2U inverted microscope</td>
			<td>Nikon</td>
			<td>MFA510BB</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Advanced Research</td>
			<td>Nikon</td>
			<td>MQS31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin conditioned media</td>
			<td>In-house</td>
			<td></td>
			<td>BMP inhibitor</td>
		</tr>
		<tr>
			<td>Pipetboy acu 2</td>
			<td>Integra</td>
			<td>155000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes (p20, p100, p1000) with tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Jomar Bioscience</td>
			<td>ant-pm2</td>
			<td>Combination of antibacterial and antifungal compounds to protect cell cultures from contaminations</td>
		</tr>
		<tr>
			<td>Prostaglandin E2 (PGE2)</td>
			<td>Tocris</td>
			<td>2296</td>
			<td>support proliferation of cells</td>
		</tr>
		<tr>
			<td>Rotary tube mixer</td>
			<td>Ratek</td>
			<td>RSM7DC</td>
			<td></td>
		</tr>
		<tr>
			<td>R-spondin 1 conditioned media</td>
			<td>In-house</td>
			<td></td>
			<td>WNT signalling regulator</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Jomar Bioscience</td>
			<td>s1077-25mg</td>
			<td>Selective p38 MAP kinase inhibitor</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Livingstone</td>
			<td>WBLDHDL03</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels, #11 blade</td>
			<td>Medical and Surgical Requisites</td>
			<td>EU-211-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes (5, 10, 25 mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Waste Bags</td>
			<td>Medical Search</td>
			<td>SU09125X16</td>
			<td></td>
		</tr>
		<tr>
			<td>Urine specimen jar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Jomar Bioscience</td>
			<td>s1049-10mg</td>
			<td>Selective ROCK inhibitor. Increases survival of dissociated epithelial cells</td>
		</tr>
	</tbody>
</table>

culture of bladder cancer organoids as precision medicine tools

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

3D organoids were successfully established from human bladder cancer patient TURBT and cystectomy tissues. Briefly, this technique highlights a rapid formation of 3D multicellular structures that are both viable and suitable for other endpoint analyses such as histological evaluation, molecular characterization (by immunohistochemistry or quantitative real-time PCR), and drug screening. During the procedure (Figure 1), the various eluates during our filtration phases (Fi...

Watch this Scientific Journal Video about Culture of Bladder Cancer Organoids as Precision Medicine Tools at JoVE.com

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Patient-derived organoids (PDOs) are a powerful tool in translational cancer research, reflecting both the genetic and phenotypic heterogeneity of the disease and response to personalized anti-cancer therapies. Here, a consolidated protocol to generate human primary bladder cancer PDOs in preparation for the evaluation of phenotypic analyses and drug responses is detailed.

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as ...

This method is significant because patient-derived organoids provide a rapid and powerful tool to study urological cancers as they frequently mimic the genetic and phenotypic heterogeneity of this multispectrum disease. Organoids can be isolated from a limited amount of patient biopsy material and can be rapidly expanded for downstream analysis. The organoids generated using this protocol can be used both as models to help us understand the cellular and molecular basis of urological cancers and as precision medicine tools to let us predict which treatments and individual patients cancer might respond to.To begin, collect a fresh macroscopically viable tumor specimen from surgery and ensure that the sample is submerged in transport medium in a sterile 50 milliliter conical tube or urine specimen jar during transit. Record specimen details including tissue weight, sample description, and details regarding any blood and urine samples on the patient specimen processing sheet. Carefully replace the transport medium with 10 milliliters of basal media and allow the tumor tissue to settle by gravity.Next, using forceps, remove the tumor tissue and place it in a sterile 90 millimeter Petri dish. Record the weight of the tissue in grams or milligrams on the clinical specimen processing sheet and dissection grid. Then using sterile forceps in a disposable scalpel blade mounted to a scalpel handle, remove the non-cancerous tissue and macroscopically visible necrotic regions.Wash the tumor pieces once or twice with cold DPBS, then collect and transfer them to a new sterile 90 millimeter Petri dish. Take a photograph, draw a tissue diagram, and plan the tissue dissection on a clinical processing grid. For histopathological analysis, place approximately 50 milligrams of the tumor tissue into a labeled disposable plastic histology cassette.Then submerge the histology cassette into a container with 5X to 10X volume of 10%neutral buffered formalin and incubate overnight. The following day, replace the formalin with 70%ethanol for storage at four degrees Celsius until the tissue can be processed. For molecular analysis, snap freeze at least one tumor piece in an RNase, DNase free 1.5 milliliter cryovial using liquid nitrogen and store at minus 80 degrees Celsius.Next, dispense five milliliters of organoid medium in the 90 millimeter Petri dish containing the remaining tumor pieces and mechanically mince the tissue as fine as possible. using a sterile number 10 scalpel blade. Transfer the finely minced tissue to a 50 milliliter conical tube and add four milliliters of organoid medium, one milliliter of 10X collagenase/hyaluronidase and 0.1 milligram per milliliter DNase I to avoid cell clumping.Incubate the minced tumor tissue in enzyme solution for one to two hours on an orbital shaker or rotator in an incubator to dissociate fragments into a cell suspension and break down collagens. Then stop the digestion by adding twice the volume of basal medium to the sample. Centrifuge the sample, aspirate, and discard the supernatant.Next, to lyse contaminating red blood cells, resuspend the pellet in five milliliters of ammonium chloride potassium buffer and incubate the tube at room temperature for three minutes or until the complete lysis of the red blood cells is seen. Then add 20 milliliters of basal medium into the tube. Centrifuge again and aspirate the supernatant.At this step, place a 10 milliliter aliquot of 2X and 1X organoid medium in a 37 degree Celsius water bath to warm. Filter the sample first through a pre-wet reversible 100 micron strainer into a new 50 milliliter tube to remove large insoluble material. Then filter the elute through a pre-wet reversible 37 micron strainer to collect single cells in small clusters for single cell and immune cell isolation.Next, reverse the 37 micron strainer and add 10 milliliters of basal media to collect small and moderate-sized clusters. Top up each of these new 50 milliliter tubes with DPBS to 40 milliliters, then centrifuge the suspension and discard the supernatant. Next, add 10 milliliters of basal media to the tube containing the single cells and count the cells using trypan blue exclusion dye in an automated cell counter.Determine the cell number and viability of cells. Centrifuge the remaining sample and replace the medium with cell freezing solution or basal media containing 10%fetal bovine serum 1%penicillin and streptomycin and 10%DMSO, then place the samples in 1.5 milliliter cryovials, store them in a self-freezing container, and immediately transfer the containers to a minus 80 degree Celsius freezer. The next day, transfer the cryovials into cryogenic liquid or air phase storage for long-term storage.Next, resuspend the collected small and moderate-sized clusters with 500 microliters of pre-warmed 2X organoid medium. Then with ice cold P-1000 sterile filter pipette tips, add BME to the cells and mix gently. Quickly and carefully pipette 100 microliters of the reconstituted cells BME mixture to wells of an ultra low attachment, flat-bottom 96-well plate, and place the plate in an incubator for 20 to 30 minutes to solidify.After incubation add organoid medium on top of the BME cell suspension in a one to two ratio depending on the empirically assessed volume, and place the plate in an incubator for 20 minutes to equilibrate. After incubation, remove the plate from the incubator and assemble it on a specimen holder on the stage of a microscope to assess organoids visually under phase contrast or bright field settings. Top up the medium every two to three days using 50 microliters of pre-warmed organoid medium to replenish depleted growth factors and overall volume.Acquire the time-lapse series images on days 1, 2, and 3, and 5, 7, and 10 before passaging. Two hour digestion of bladder cancer tissue with commercially available collagenase/hyaluronidase and DNase I leads to sufficient digestion of 0.5 to 1 cubic millimeter pieces of more complex cystectomy biopsy tissue, including the neoplastic cells within the lamina propria and muscular layers of the bladder. Larger post-digestion fragments are suitable for culture and standard tissue culture plates of any size to isolate novel two dimensional cultures.Organoids generated by this procedure undergo dynamic self-assembly, including phases of aggregation, compaction, and final formation of tight cellular structures. Histologically, these structures recapitulate the original patient tumor. Organoids generated in this procedure are suitable for many purposes, including further histopathological characterization, biobanking, and drug efficacy testing.Following generation of organoids, additional methods can be used to profile these tumors, including the role of anti-cancer therapies, metabolic profiling, or transcription profiling. Within this 3D framework, these additional methodologies provide powerful insight into bladder cancer biology. This method was an important foundation for studies investigating immunooncology and cellular metabolism.

culture-of-bladder-cancer-organoids-as-precision-medicine-tools

Research

JoVE Journal

Cancer Research

4.2K Görüntüleme.  Queensland University of Technology (QUT) at Translational Research Institute. Bu yöntem önemlidir, çünkü hasta kaynaklı organoidler, bu çok spektrumlu hastalığın genetik ve fenotipik heterojenliğini sıklıkla taklit ettikleri için ürolojik kanserleri incelemek için hızlı ve güçlü bir araç sağlarlar. Organoidler sınırlı miktarda hasta biyopsi materyalinden izole edilebilir ve aşağı akış analizi için hızla genişletilebilir. Bu protokol kullanılarak üretilen organoidler, hem ürolojik kanserlerin hücresel ve moleküler temelini anlamam?...